Sho Konno

Thioesterase (TE) domains catalyze peptide release and macrocyclization during nonribosomal peptide biosynthesis, typically via catalytic serine. In contrast, MacB-TE from macolacin biosynthesis employs a rare cysteine nucleophile, raising questions regarding the catalytic requirements of cysteine-dependent peptide cyclases. Here, we report the biochemical and functional characterization of MacB-TE and demonstrate its efficiency, regioselectivity, and exclusive macrocyclization. Using a synthetic thioester substrate, MacB-TE converted >99% of the linear precursor into the cyclic product within minutes. Mutational analysis demonstrated that Cys89 is indispensable for ring formation, whereas His218 and Asp116 support an efficient turnover. Notably, a D116S variant retained substantial cyclization activity, uncovering an unexpected tolerance at the acidic position of the catalytic triad. Comprehensive substrate profiling showed that MacB-TE accommodated extensive side-chain variations and diverse N-terminal lipid groups. The enzyme efficiently macrocyclized all nine alanine-scan variants, with six positions supporting near-quantitative conversion and the remaining three producing more than 80% of the cyclic products. Likewise, lipid-modified substrates, including those with aliphatic and aromatic motifs, were quantitatively converted into their corresponding macrocycles, indicating that lipid variation had a negligible impact on catalysis. Collectively, these findings establish MacB-TE as a highly efficient and broadly tolerant macrocyclase, enabling the chemoenzymatic synthesis of macolacin analogs and expanding the mechanistic understanding of Cys-type TEs.

Gramicidin S (GS) is a cyclic decapeptide derived from two pentapeptides. The C‐terminal thioesterase (TE) domain of gramicidin S synthetase B (GrsB) dimerizes precursor pentapeptides and cyclizes the resulting linear decapeptide. Recently, a GS variant (GS‐SA), in which a single D‐Phe is replaced by L‐Ser(Allyl), is reported via precursor‐directed biosynthesis in a native GS producer. To understand how GrsB‐TE processes such modified precursors, its substrate specificity using synthetic linear peptides is investigated. GrsB‐TE cyclizes a substrate containing L‐Ser(Allyl) at position 6 but not at position 1. However, the enzymatically synthesized GS‐SA shows a different high‐performance liquid chromatography retention time than that of the reported GS variant. Further structural and functional analyses, including ¹ H nuclear magnetic resonance, antimicrobial assays, and circular dichroism spectroscopy, reveal that the reported GS‐SA contained D‐Ser(Allyl) rather than L‐Ser(Allyl). These findings reveal a previously unrecognized stereochemical flexibility in GrsB‐TE and support the structural revision of the reported GS variant.

Establishing an efficient method for the synthesis of disulfide bonds in peptides is an important challenge for the further development of several research fields, including peptide‐based medicinal chemistry and biochemistry. Herein, we report the development of a novel highly water‐soluble 3‐nitro‐2‐pyridine (Npy) sulfenate that efficiently forms intramolecular disulfide bonds in reduced peptides. Moreover, Npy‐sulfenate formed a disulfide bond in a thiol‐containing peptide prepared by thiol‐additive‐free native chemical ligation (NCL), as demonstrated by the one‐pot synthesis of adrenomedullin, which contains 52 amino acid residues. This study provides a new one‐pot synthetic methodology for preparing mid‐sized cyclic disulfide peptides via sulfenate‐mediated oxidation.

Several natural bacterial virulence factors are biosynthesized by nonribosomal peptide synthetases (NRPSs). Therefore, NRPSs producing such natural products have emerged as attractive antibiotic targets. N‐(aminoacyl)sulfamoyladenosine (aminoacyl‐AMS) derivatives with chemical modifications at 2′‐OH group of the adenosine skeleton are previously reported that inhibit NRPS amino acid adenylation (A) domains in recombinant enzyme systems, cellular lysates, and bacterial cells. The introduction of a couple of functionalities at the 2′‐OH group preserves the binding affinity toward NRPS amino acid A‐domains and improves cell permeability of the AMS scaffold. However, the effects of these compounds on secondary metabolism have not yet been explored. In this study, an affinity‐based protein profiling (AfBPP) probe, L‐Leu‐AMS‐BPyne, is validated for in‐cell applications, including imaging of NRPS activities in bacteria. Next, L‐Leu‐AMS derivatives incorporating methyl, benzyl, and cyanomethyl functionalities at the 2′‐OH group are synthesized and their inhibitory activity toward intracellular surfactin‐NRPSs in the surfactin‐producer Bacillus subtilis ATCC 21332 are investigated using the AfBPP probe. Finally, the attenuation of surfactin production using a Leu‐AMS‐BPyne probe and L‐Leu‐AMS derivatives in B. subtilis is demonstrated. These results indicate that chemical modifications at the 2′‐OH group provide a way to develop cell‐permeable and functional NRPS A‐domain inhibitors.

Chemical modification of biomolecules such as peptides and proteins is an important technology for the development of new functional molecules. Herein, a thioether‐mediated sulfenylation for tryptophan (Trp)‐selective chemical modification under mild metal‐free reaction conditions involving a weak acid/water solvent is reported. An electrophilic species generated from 4‐fluorophenyl 3‐nitro‐2‐pyridinesulfenate, Npys‐OPh(pF), in the presence of a thioether component, enables efficient sulfenylation of the Trp residue on various Trp‐containing biologically active linear and cyclic peptides. In addition, this sulfenylation to late‐stage chemical modification of a natural cyclic depsipeptide, daptomycin, is expanded and Trp‐sulfenylated derivatives, including fluorescent‐labeled derivatives, are obtained. The results indicate that the thioether‐mediated sulfenylation can be used as a robust Trp‐selective chemical modification method for peptides in a wide range of applications.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5’-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2′-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2′-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS‐TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate‐TE interaction during the macrocyclization step are still unknown. To understand the TE‐mediated macrocyclization, here we report the development of a substrate‐based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p‐nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)‐TE containing tyrocidine synthetase.

A revised structure of natural 14‐mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid‐phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell‐based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin‐1 is a key to new avenues for transdermal administration of large hydrophilic biologics.